Characterization of the nucleoside monophosphate kinase from <i>Thermus thermophilus</i> phage p23-45 and its application in the biosynthesis of deoxynucleoside triphosphates

Nucleoside monophosphate kinases (NMKs) are essential enzymes in nucleotide biosynthesis. In this study, an NMK from Thermus thermophilus phage p23-45 (ϕNMK) was overexpressed and characterized, which exhibited relatively low sequence similarity (∼20%) to cellular NMKs. The enzyme demonstrated broad specificity for all 4 deoxynucleoside monophosphates (dNMPs) (KM: 0.27-0.45 m m), optimal activity at 70 °C, and retained 95% activity after 8 h at 37 °C. Structural modeling and site-directed mutagenesis identified key catalytic roles for K39 and R66. A one-pot deoxyribonucleoside triphosphate (dNTP) synthesis workflow using ϕNMK and T. thermophilus pyruvate kinase enhanced ATP regeneration efficiency compared to acetate kinases, achieving >85% conversion of all dNMPs to dNTPs. These findings highlight ϕNMK's thermostability and efficiency, establishing it as a promising candidate for industrial dNTP production.